Characterization of Escherichia coli Gene Mutations that Affect Growth During Metabolic Stress
Mentor 1
Gregory Richards
Location
Union Wisconsin Room
Start Date
24-4-2015 10:30 AM
End Date
24-4-2015 11:45 AM
Description
Glucose-phosphate stress, a metabolic condition that affects bacteria such as Escherichia coli, occurs when too much glucose-6-phosphate (G6P) or other related sugar-phosphates accumulate in the cell and inhibit growth. The small regulatory RNA (sRNA) SgrS and the transcriptional regulator SgrR have been identified in E. coli as important regulators that help the cell recover from this stress. SgrR activates transcription of the sgrS gene, and SgrS works by blocking mRNA translation of genes associated with sugar metabolism and transport like ptsG, which encodes the major glucose transporter. While regulation by SgrS has been extensively characterized, very few regulatory targets of SgrR are currently known. The goal of our research is to identify additional genes regulated by SgrR and characterize their role during glucose-phosphate stress. Our lab has previously identified putative gene targets of SgrR by using high-throughput RNA sequencing analysis (RNA-seq) to compare whole-genome expression in wild-type E. coli and an sgrR deletion mutant grown under stress conditions. To begin to characterize the relevance of these putative SgrR targets to stress, we introduced several target gene insertion-deletion mutations into wild-type and sgrS mutant backgrounds using P1 phage transduction and screened the strains for changes in growth during glucose-phosphate stress. Genes encoding functions related to sugar transport and metabolism that showed at least a 2-fold expression difference between wild-type and the sgrR mutant were prioritized. Several mutants tested to date did not exhibit significant growth changes during stress. However, preliminary results indicate that mutating fbp, which encodes a fructose-1,6-bisphosphatase required to synthesize glucose, results in a slight increase in growth compared to the wild-type parent during stress. Current directions include characterizing stress response induction in the fbp mutant, as well as constructing additional mutations in metabolism-related genes and analyzing the mutants for stress-related phenotypes.
Characterization of Escherichia coli Gene Mutations that Affect Growth During Metabolic Stress
Union Wisconsin Room
Glucose-phosphate stress, a metabolic condition that affects bacteria such as Escherichia coli, occurs when too much glucose-6-phosphate (G6P) or other related sugar-phosphates accumulate in the cell and inhibit growth. The small regulatory RNA (sRNA) SgrS and the transcriptional regulator SgrR have been identified in E. coli as important regulators that help the cell recover from this stress. SgrR activates transcription of the sgrS gene, and SgrS works by blocking mRNA translation of genes associated with sugar metabolism and transport like ptsG, which encodes the major glucose transporter. While regulation by SgrS has been extensively characterized, very few regulatory targets of SgrR are currently known. The goal of our research is to identify additional genes regulated by SgrR and characterize their role during glucose-phosphate stress. Our lab has previously identified putative gene targets of SgrR by using high-throughput RNA sequencing analysis (RNA-seq) to compare whole-genome expression in wild-type E. coli and an sgrR deletion mutant grown under stress conditions. To begin to characterize the relevance of these putative SgrR targets to stress, we introduced several target gene insertion-deletion mutations into wild-type and sgrS mutant backgrounds using P1 phage transduction and screened the strains for changes in growth during glucose-phosphate stress. Genes encoding functions related to sugar transport and metabolism that showed at least a 2-fold expression difference between wild-type and the sgrR mutant were prioritized. Several mutants tested to date did not exhibit significant growth changes during stress. However, preliminary results indicate that mutating fbp, which encodes a fructose-1,6-bisphosphatase required to synthesize glucose, results in a slight increase in growth compared to the wild-type parent during stress. Current directions include characterizing stress response induction in the fbp mutant, as well as constructing additional mutations in metabolism-related genes and analyzing the mutants for stress-related phenotypes.
